1. Field of the Invention
The present invention generally relates to nucleic acid molecules derived from equine encephalitis viruses and compositions and methods thereof.
2. Description of the Related Art
Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV) are non-segmented, positive-sense RNA viruses of the genus Alphavirus in the family Togaviridae. See Griffin (2001) “Alphaviruses”, p. 917-962, in FIELDS VIROLOGY, vol. 4. Lippincott, Williams, and Wilkins, Philadelphia, Pa. Naturally transmitted by mosquitoes through rodent or bird hosts, VEEV, EEEV, and WEEV are highly pathogenic for equines and humans and have caused periodic epizootics throughout North, Central, and South America. See Tsai (1991) Infect Dis Clin North Am 5:73-102. Human infection with these New World alphaviruses typically results in an acute, incapacitating disease characterized by fever, headache, lymphopenia, myalgia, and malaise. See Bale (1993) Med Clin North Am 77:25-42. Severe neurological disease, including fatal encephalitis, can also result from VEEV, EEEV, and WEEV infection of humans. Although the human case-fatality rates are estimated to be low for VEEV (≦1%) and WEEV (8-15%), EEEV is the most severe of the arbovirus encephalitides with a human case-fatality rate estimated to be 30-70%. See Steele et al. (2007) “alphavirus Encephalitides” p. 241-270, in MEDICAL ASPECTS OF BIOLOGICAL WARFARE. BORDEN INSTITUTE (U.S. Army Walter Reed), Washington, D.C. However, numerous documented laboratory accidents and the results of animal studies have demonstrated that VEEV, EEEV, and WEEV are also highly infectious in aerosols, and infection with aerosolized virus could potentially result in higher mortality than that observed with natural infection. See Franz et al. (2001) Clin Lab Med 21:435-73; Hanson (1967) Science 158:1283-6; and Kortepeter et al. (2001) J Environ Health 63:21-4. In addition to producing incapacitating or lethal infections and being infectious in aerosols, these encephalitic alphaviruses are also easily grown to high titers in inexpensive and unsophisticated cell culture systems and are relatively stable. As a result, VEEV, EEEV, and WEEV represent significant potential biological defense threats and are classified as Category B priority biodefense agents by both the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases.
Although there are no licensed human vaccines for the encephalitic alphaviruses, live-attenuated and formalin-inactivated vaccines are currently being utilized under Investigational New Drug (IND) status to protect laboratory workers and other at-risk personnel. A live-attenuated vaccine for VEEV, TC-83, provides long-lasting immunity and protection from both subcutaneous and aerosol VEEV challenges; however, it causes adverse reactions in approximately 25% of recipients, and approximately 20% of recipients fail to develop a detectable neutralizing antibody response. See McKinney et al. (1963) Am J Trop Med Hyg 12:597-603; and Pittman et al. (1996) Vaccine 14:337-43. C-84 (formalin-inactivated TC-83 VEEV vaccine), and EEEV and WEEV formalin-inactivated vaccines are well tolerated, but they require frequent boosting to elicit detectable neutralizing antibody responses in humans and have provided poor protection against aerosol viral challenge in animal studies. See Cole et al. (1973) Appl Microbiol 25:262-5; Bartelloni et al. (1970) Am J Trop Med Hyg 19:123-6; and Bartelloni et al. (1971) Am J Trop Med Hyg 20:146-9. Due to the significant limitations associated with the existing live-attenuated and formalin-inactivated vaccines currently being utilized under IND status, the development of improved vaccines that can safely and effectively protect against encephalitic alphavirus infections in humans is needed.
Next-generation VEEV vaccines, including live-attenuated, inactivated, attenuated Sindbis/VEEV chimeric viruses, alphavirus replicons, and DNA vaccines, are all currently at various stages of development. See Paessler & Weaver (2009) Vaccine 27 Suppl 4:D80-5. Genetic vaccination with DNA plasmids expressing immunogenic proteins has numerous inherent advantages as a platform for the development of next-generation vaccines. Among the benefits of this method are that DNA vaccines can be rapidly and cost-effectively produced without the need to propagate a pathogen, do not require the inactivation of infectious organisms, avoid problems of preexisting or vector-induced immunity due to lack of a host immune response to the plasmid backbone, and have exhibited a favorable safety profile in numerous human clinical trials. See Dupuy & Schmaljohn (2009) Expert Rev Vaccines 8:1739-54.
In previous studies, mice vaccinated with a DNA vaccine expressing the structural proteins (C-E3-E2-6K-E1) of VEEV subtype IAB (strain Trinidad Donkey) by particle-mediated epidermal delivery (PMED) or “gene gun”, in which plasmid DNA-coated gold particles are delivered intradermally in a ballistic manner, developed strong overall antibody responses against VEEV IAB. Unfortunately, the VEEV-neutralizing antibody responses were low, and only 80% protection against lethal aerosol challenge was observed. See Riemenschneider et al. (2003) Vaccine 21:4071-80. Cynomolgus macaques vaccinated with this VEEV DNA vaccine by PMED developed detectable levels of VEEV IAB-neutralizing antibodies, but only partial protection was observed upon aerosol challenge. See Dupuy et al. (2010) Vaccine 28:7345-50.
In other studies to develop a human vaccine for encephalitic alphaviruses, directed molecular evolution or “gene shuffling” of the envelope protein genes was used as an attempt to improve the neutralizing antibody response to VEEV, EEEV, and WEEV DNA vaccines. DNA vaccines expressing representative variants from a library in which the E2 envelope glycoprotein genes of five parent viruses (VEEV subtypes IAB and IE, Mucambo virus, EEEV (strain PE6), and WEEV (strain CBA87) were recombined and the E1 envelope glycoprotein gene of VEEV IAB was held constant elicited significantly increased neutralizing antibody titers to VEEV IAB compared to the wild-type parent VEEV DNA vaccine and provided improved protection against aerosol VEEV IAB challenge in mice. See Dupuy et al. (2009) Vaccine 27:4152-60. Unfortunately, in addition to the in vitro gene recombination being technically difficult and the screening of variants for improved immunogenicity being labor-intensive, the studies failed to result in variant envelope glycoprotein vaccines having improved immunogenicity against EEEV and WEEV as compared to the wild-type parent EEEV and WEEV DNA vaccines.
Therefore, a need still exists for safe and effective vaccines to protect against equine encephalitis viruses (EEVs) such as VEEV, EEEV, and WEEV.